The present invention relates to a lipoprotein adsorbent. More particularly, it relates to a lipoprotein adsorbent for selectively adsorbing and removing lipoproteins, including apoB proteins, in particular low density lipoproteins (hereinafter, LDL) and very low density lipoproteins (hereinafter, VLDL), from blood constituents or the like.
Lipoproteins occurring in blood, in particular LDL and VLDL, commonly known as a villain cholesterol, contain a large amount of cholesterol and are known to be causative of atherosclerosis. On the other hand, the high density lipoprotein (hereinafter, HDL), commonly known as a goody cholesterol, is known to serve as an atherosclerosis retarding factor. Therefore, means is desired by which LDL and VLDL can be removed from blood constituents or the like without removing HDL therefrom.
As the methods currently in clinical use for removing LDL and VLDL, there can be mentioned plasma exchange therapy, double filtration membrane method, use of adsorbents (immobilized dextran sulfate, immobilized anti-apoB antibody, etc.) and HELP system, among others.
However, the membrane method brings about simultaneous removal of a considerable amount of HDL together with LDL and VLDL and therefore fails to satisfy the selectivity for lipoprotein. Further, there is a drawback that plasma proteins are partly removed simultaneously, hence such losses need to be repaired by supplementation.
As the removing method using an adsorbent, there can be mentioned, for example, the removing method using the so-called immunoadsorbent comprising an antibody or the like immobilized and the removing method using an adsorbent comprising a compound having affinity for LDL and VLDL (hereinafter, ligand) as immobilized according to the principle of the so-called affinity chromatography.
The removing method using an immunoadsorbent, however, has problems although it satisfies the selectivity for lipoprotein; thus, for example, the antibody to be used is not readily available or is poor in economy or storage stability or is difficult to sterilize.
In the removing method which uses an adsorbent based on the principle of affinity chromatography, heparin, dextran sulfate and the like are used as ligands of adsorbents. The adsorbents in which these ligands are used show good selectivity for lipoprotein and the ligands themselves are not very expensive. In cases where a ligand is used in large amounts, however, it is desired that said ligand is less expensive.
As an adsorbent for adsorbing and removing lipoproteins by using such an organic compound as phenyl glycidyl ether as a ligand and utilizing the hydrophobic interaction between the phenyl group and the hydrophobic sites of the lipoprotein surface, there is commercially available Phenyl Sepharose CL-4B (product of Pharmacia Fine Chemicals). However, this adsorbent, though it is inexpensive, adsorbs large amounts of HDL as well as LDL and VLDL and thus has a serious drawback from the standpoint of the selectivity for lipoprotein.
In view of the above-mentioned state of the art, the present invention has its objects to provide an adsorbent capable of efficiently adsorbing and removing LDL and VLDL from various lipoprotein-containing solutions, such as blood, serum, plasma, dilutions thereof, and solutions resulting from pretreatment of them for removal of blood corpuscles or serum protein or the like as well as a lipoprotein adsorber in which said adsorbent is used.
Japanese Kokai Publication Sho-62-186940 describes a lipoprotein adsorbent which comprises a water-insoluble carrier having aniline or an aniline derivative immobilized thereon and, in Japanese Kokai Publication Sho-63-208764, there is described a lipoprotein adsorbent comprising a water-insoluble matrix and, on the surface thereof, a group represented by the general formula xe2x80x94NRaRb in which Rb is a group such that the corresponding compound RbH has a logarithmic value (log P) of 0 to 3.2, P being the partition coefficient in the water-octanol system, and it is disclosed that an inexpensive lipoprotein removing apparatus capable of removing LDL and VLDL selectively can be provided by using said adsorbent.
Referring to these technologies, the present inventors made further investigations and, as a result, found that when the lipoprotein adsorbents having a group represented by the above general formula xe2x80x94NRaRb further have a substituent in the meta position of Rb, which is the aromatic ring-containing atomic group in said group xe2x80x94NRaRb, the selectivity for LDL and VLDL, in particular, is high, that when, in cases where Rb is a group bound to an aromatic ring via an atom or atomic group, it is bound to the aromatic ring via a carbonyl group, the selectivity for LDL and VLDL is high; and that when Rb has a specific functional group, the selectivity for LDL and VLDL is particularly high.
Thus, the present invention provides a lipoprotein adsorbent comprising a water-insoluble carrier having, on at least one part of the surface of said carrier, at least one group (other than p-nitrobenzoic acid) selected from the group consisting of groups represented by the general formula 
(hereinafter also represented as xe2x80x9cxe2x80x94NR1xcfx861Xxe2x80x9d) (wherein R1 represents a hydrogen atom or a methyl or ethyl group, xcfx861 represents an atomic group comprising an aromatic ring bound directly or via one atom to the nitrogen atom, X represents a substituent atom or atomic group bound to a meta position of said aromatic ring, and xcfx861X represents an atomic group such that the compound represented by Hxcfx861X has a logP value (P being the partition coefficient in the water-octanol system) of 0 to 3.2), and groups represented by the general formula 
(hereinafter also represented as xe2x80x9cxe2x80x94NR2COxcfx862Yxe2x80x9d) wherein R2 represents a hydrogen atom or a methyl or ethyl group, CO represents a carbonyl group, xcfx862 represents an atomic group comprising an aromatic ring bound to the nitrogen atom via said carbonyl group, Y represents an atomic group bound to said aromatic ring, to the exclusion of the case where Hxcfx862Y is benzene, and COxcfx862Y represents an atomic group such that the compound represented by Yxcfx862COH has a logP value of 0 to 3.2 (P being the partition coefficient in the water-octanol system). The present invention further provides a lipoprotein adsorber which comprises an adsorption section containing the lipoprotein adsorbent mentioned above, a liquid inlet section for a liquid to flow into said adsorption section and a liquid discharge section for the liquid that has flown into said adsorption section to flow out of said adsorption section.
In the following, the present invention is described in detail.
The lipoprotein adsorbent of the present invention has, on at least one part of the surface of a water-insoluble carrier, at least one group selected from the group consisting of a group represented by the general formula xe2x80x94NR1xcfx861X (wherein R1, xcfx861 and X are as defined above) and a group represented by the general formula xe2x80x94NR2COxcfx862Y (wherein R2, CO, xcfx862 and Y are as defined above).
In the above group xe2x80x94NR1xcfx861X, R1 represents a hydrogen atom, methyl group or ethyl group. The above-mentioned xcfx861 represents an atomic group comprising an aromatic ring bound to the nitrogen atom either directly or via an atom. The above-mentioned X represents an atomic group bound to the meta position of said aromatic ring. Further, xcfx861X resulting from binding xcfx861 and X to each other is such that the compound represented by Hxcfx861X has a logP value of 0 to 3.2 (P being the partition coefficient in the water-octanol system).
The logP value, which is the logarithmic value of the partition coefficient in the water-octanol system, is a parameter indicative of the degree of hydrophobicity of a compound. A typical method of determining the partition coefficient P is as follows.
The compound is dissolved in octanol (or water), an equal volume of water (or octanol) is added to the solution, and the mixture is shaken on a Griffin flask shaker (product of Griffin and George Limited) for 30 minutes and centrifuged at 2,000 rpm for 1 to 2 hours. The concentrations of the compound in the octanol layer and aqueous layer are determined by any of various methods, for example by spectrophotometry or GLC (gas-liquid chromatography), and the P value is calculated as follows:
P=Coct/Cwxe2x80x83xe2x80x83(1)
where Coct is the concentration of the compound in the octanol layer and Cw is the concentration of the compound in the aqueous layer.
In accordance with the present invention, the logP value for the compound represented by Hxcfx861X is 0 to 3.2. If the logP value is less than 0, the hydrophobic interaction with lipoproteins will be weak, hence the adsorbing ability for lipoprotein will be poor. If said logP value exceeds 3.2, not only LDL and VLDL but also HDL and other proteins are simultaneously adsorbed, hence a problem from the selectivity viewpoint will arise. The above range is thus critical. It is preferred that said logP value be 0.8 to 2.7. While it is stated in Japanese Kokai Publication Sho-63-208764 that the hydrophobicity of xcfx861X in the group xe2x80x94NR1xcfx861X mentioned above plays an important role in the adsorption of lipoproteins, it has now newly been found in the present invention that, in addition to the degree of hydrophobicity, the position of the functional group bound to the aromatic ring is related to the adsorptivity for lipoproteins and that when the adsorbent has the functional group in the meta position, it shows particularly high selectivity as compared with those having the functional group in the ortho or para position.
Said logP value may vary to some extent but not to a great extent depending on the position (ortho, meta, para) of the substituent functional group on the aromatic ring, so that said logP value may be the same as in the case of Japanese Kokai Publication Sho-63-208765.
The term xe2x80x9cselectivexe2x80x9d in this specification means that the adsorptivity of HDL for the adosorbent is low and the adsorptivity for LDL and VLDL is high. The value calculated according to the following formula (2) is used as an index of selectivity.
Index of selectivity=(% adsorption of HDL)/(% adsorption of LDL)xe2x80x83xe2x80x83(2)
The nearer to zero the value of the above formula (2) is, the higher the selectivity is.
The aromatic ring contained in the above-mentioned group xcfx861 is not particularly restricted but includes, among others, benzene, pyridine, pyrimidine and triazine rings and condensed rings derived from these.
The above-mentioned compound represented by Hxcfx861X is not particularly restricted but may be any compound having a logP value of 0 to 3.2. Thus, for example, when the aromatic ring contained therein is a benzene ring, there may be mentioned toluene, m-xylene, ethylbenzene, phenol, benzyl alcohol, 2-phenylethanol, benzaldehyde, anisole, phenetole, phenylacetic acid, phenoxyacetic acid, methyl benzoate, nitrobenzene, chlorobenzene, bromobenzene, fluorobenzene, m-dinitrobenzene, 3-nitrobenzaldehyde, 3-nitroanisole, 3-nitrotoluene, benzamide, acetophenone, 3-ethylphenol, 3-ethoxyphenol, acetanilide, 3-methylbenzyl alcohol and the like.
When the aromatic ring contained in said xcfx861is a pyridine ring, as said Hxcfx861X, there can be mentioned, for example, 2-amino-4-fluoropyridine, 2-amino-6-fluoropyridine, 3-amino-5-fluoropyridine, 4-amino-6-fluoropyridine, 2-amino-4-nitropyridine, 2-amino-6-nitropyridine, 3-amino-5-4-nitropyridine, 4-amino-6-nitropyridine, 2-amino-4-hydroxypyridine, 2-amino-6-hydroxypyridine, 3-amino-5-hydroxypyridine, 4-amino-6-hydroxypyridine, 2-amino-4,6-difluoropyridine, 2-amino-4,6-dinitropyridine, 2-amino-4,6-dihydroxypyridine, 4-amino-2,6-difluoropyridine, 4-amino-2,6-dinitropyridine, 4-amino-2,6-dihydroxypyridine, 4-fluoropyridine-2-carboxamide, 6-fluoropyridine-2-carboxamide, 4-nitropyridine-2-carboxamide, 6-nitropyridine-2-carboxamide, 4-hydroxypyridine-2-carboxamide, 6-hydroxypyridine-2-carboxamide, 2-carboxy-4-fluoropyridine, 2-carboxy-6-fluoropyridine, 2-carboxy-4-nitropyridine, 2-carboxy-6-nitropyridine, 2-carboxy-4-hydroxypyridine, 2-carboxy-6-hydroxypyridine and the like.
In cases where the aromatic ring contained in the above xcfx861 is a pyrimidine ring, there may be mentioned, as examples of said compound Hxcfx861X, 2-amino-4-fluoropyrimidine, 2-amino-6-fluoropyrimidine, 4-amino-2-fluoropyrimidine, 4-amino-6-nitropyrimidine, 2-amino-6-nitropyrimidine, 2-amino-4-hydroxypyrimidine, 2-amino-6-hydroxypyrimidine, 4-amino-6-hydroxypyrimidine, 2-amino-4,6-difluoropyrimidine,2-amino-4,6-dinitropyrimidine, 2-amino-4,6-dihydroxypyrimidine, 4-amino-2,6-difluoropyrimidine, 4-amino-2,6-dinitropyrimidine, 4-amino-2,6-dihydroxypyrimidine 2-carboxy-4-fluoropyrimidine, 2-carboxy-6-fluoropyrimidine, 4-carboxy-2-fluoropyrimidine, 4-carboxy-6-nitropyrimidine, 2-carboxy-6-nitropyrimidine, 2-carboxy-4-hydroxyprimidine, 2-carboxy-6 hydroxypyrimidine, 4-carboxy-6-hydroxypyrimidine and the like.
In cases where the aromatic ring contained in the above xcfx861 is a triazine ring, there may be mentioned, as examples of said compound Hxcfx861X, 2-amino-4,6-difluoro-1,3,5-triazine, 2-amino-4,6-dichloro-1,3,5-triazine, 2-amino-4,6-dinitro-1,3,5-triazine, 2-amino-4,6-dihydroxy-1,3,5-triazine, 2-carboxy-4,6-difluoro-1,3,5-triazine, 2-carboxy-4,6-dichloro-1,3,5-triazine, 2-carboxy-4,6-dinitro-1,3,5-triazine, 2-carboxy-4,6-dihydroxy-1,3,5-triazine, 3-amino-5-fluoro-1,2,4-triazine, 3-amino-5-bromo-1,2,4-triazine, 3-amino-5-chloro-1,2,4-triazine, 3-amino-5-nitro-1,2,4-triazine, 3-amino-5-hydroxy-1,2,4-triazine, 3-carboxy-5-fluoro-1,2,4-triazine, 3-carboxy-5-bromo-1,2,4-triazine, 3-carboxy-5-chloro-1,2,4-triazine, 3-carboxy-5-nitro-1,2,4-triazine, 3-carboxy-5-hydroxy-1,2,4-triazine and the like.
Referring to the above-mentioned group xe2x80x94NR1xcfx861H, from the ligand structure viewpoint, those ligands in which the functional group bound to the aromatic ring is bound to the meta position thereof show higher selectivity for LDL than those ligands in which the functional group is bound to the ortho or para position.
The xe2x80x9cmeta positionxe2x80x9d in this specification means the meta position for the aromatic ring of the corresponding xe2x80x94NR1xcfx861H in regard to said xe2x80x94NR1xcfx861X group occurring in the adsorbent of the present invention (thus, in the case of a benzene ring, position 3 or position 5).
The above-mentioned X may be bound to either one of the meta positions of said aromatic ring or both meta positions simultaneously. Further, in the case of the above-mentioned X bound to both meta positions simultaneously, the kind of the above-mentioned X may be the same or different.
Said X is not particularly restricted but includes, for example, nitro, hydroxy, fluoro, bromo, chloro, iodo, methyl, ethyl, methoxy, ethoxy, thiol, cyano, amino, acetyl, hydrazyl, carboxy, isocyanato, isothiocyanato, aldehyde group and the like. In cases where said X can have another substituent, said X may have a further substituent. Among the substituents specifically mentioned above, halogens, and hydroxy, nitro, acetyl, thiol and aldehyde groups are preferred.
The xe2x80x94NR1xcfx861X group has been described above. More specifically, as HNR1xcfx861X corresponding to the compound having the above-mentioned group xe2x80x94NR1xcfx861X, there can be mentioned 3-5-difluoroaniline, m-hydroxybenzylamine, m-nitrobenzylamine, m-fluorobenzylamine, m-hydroxybenzhydrazide, m-nitrobenzhydrazide, m-fluorobenzhydrazide, m-hydroxythiobenzamide, m-nitrothiobenzamide, m-fluorothiobenzamide, m-hydroxybenzenesulfonamide, m-nitrobenzenesulfonamide, m-fluorobenzenesulfonamide, m-hydroxyaniline, m-nitroaniline, m-fluoraniline, m-mercaptoaniline, m-methoxyaniline, m-aminophenol, m-aminoacetophenone, m-aminoindazole, m-aminobenzyl alcohol, m-hydroxybenzoic acid, m-nitrobenzoic acid, m-fluorobenzoic acid and the like. Among them, 3,5-difluoroaniline, m-aminophenol, m-aminoacetophenone, m-nitroaniline and m-hydroxybenzoic acid are preferred. These may be used singly or two or more of them may be used in combination.
When the lipoprotein adsorbent of the present invention has the above-mentioned group xe2x80x94NR1xcfx861X, selectivity for that is improved, namely the lipoprotein adsorbent has at least about 1.5 times and at most about 7 times higher selectivity when compared, in terms of the selectivity index defined by the above formula (2), with the lipoprotein adsorbents disclosed in Japanese Kokai Publication Sho-63-208764.
The aromatic group of the xcfx861 mentioned above is bound to the nitrogen atom of the group xe2x80x94NR1xcfx861X directly or via an atom. In cases where it is bound via an atom, such an atom or atomic group may be a carbon, nitrogen, oxygen, sulfur or phosphorus atom or the like. The nitrogen atom in the above group xe2x80x94NR1xcfx861X is associated with the selectivity difference between LDL and HDL. According to the method of introducing the xe2x80x94NR1xcfx861X group into the water-insoluble carrier, the nitrogen atom in said group xe2x80x94NR1xcfx861X may be one originating from the water-insoluble carrier or one originating from the ligand. For example,
(1) when said xe2x80x94NR1xcfx861X group is a group derived from a compound represented by the general formula 
xe2x80x83(in which R1, xcfx861 and X are as defined above) (hereinafter also expressed as xe2x80x9cHNR1xcfx861Xxe2x80x9d) and is immobilized on the water-insoluble carrier via the nitrogen atom of the above-mentioned compound, the nitrogen atom in said xe2x80x94NR1xcfx861X group is of ligand origin and
(2) when said xe2x80x94NR1xcfx861X group is a group introduced into a water-insoluble carrier by reacting the water-insoluble carrier having a group represented by the general formula 
xe2x80x83(in which R1 is as defined above) (hereinafter also expressed as xe2x80x9cxe2x80x94NR1Hxe2x80x9d) with a compound represented by the general formula Zxcfx861X (in which X and xcfx861 and X are as defined above and Z represents a functional group capable of reacting with an amino group or a part of said functional group) the nitrogen atom in said xe2x80x94NR1xcfx861X group is of the water-insoluble carrier origin.
Preferred as the compound represented by the above formula HNR1xcfx861X are compounds in which X is a group selected from the group consisting of halogens and hydroxy, nitro, acetyl, thiol and aldehyde groups, and mixtures thereof. The compound represented by the above formula HNR1xcfx861X may be an aniline derivative or a mixture of aniline derivatives, a benzylamine derivative or a mixture of benzylamine derivatives, or the like. These are readily available, hence are particularly useful. The above-mentioned aniline derivative is not particularly restricted but includes, for example, aromatic alkyl-substituted anilines such as m-toluidine and 3,5-xylidine; aromatic alkoxy-substituted anilines such as m-aminoanisole and 3-aminophenetole; anilines having one or more substituents of one or more kinds on the aromatic ring thereof, such as m-chloroaniline, m-bromoaniline, m-fluoroaniline, m-nitroaniline, 3,5-dinitroaniline, 3,5-dichloroaniline, 3,5-dibromoaniline, 3,5-difluoroaniline, m-aminobenzoic acid, ethyl m-aminobenzoate, 3-aminoacetophenone, m-aminophenol, m-aminothiophenol, m-aminophenethyl alcohol, m-aminobenzyl alcohol and m-phenylenediamine; and the like. Among them, 3,5-difluoroaniline, m-aminophenol, 3-aminoacetophenone and m-nitroaniline are preferred. These may be used singly or two or more of them may be used in combination.
Said benzylamine derivative is not particularly restricted but includes, for example, meta-fluorobenzylamine, 3,5-difluorobenzylamine, meta-bromobenzylamine, 3,5-dibromobenzylamine, meta-iodobenzylamine, 3,5-diiodobenzylamine, meta-chlorobenzylamine, 3,5-dichlorobenzylamine, meta-nitrobenzylamine, 3,5-dinitrobenzylamine, meta-hydroxybenzylamine, 3,5-dihydroxybenzylamine, meta-mercaptobenzylamine, 3,5-dimercaptobenzylamine and the like.
The compound represented by the above formula Zxcfx861X is not particularly restricted but includes, for example, m-hydroxybenzamide, m-nitrobenzaldehyde, m-methoxybenzaldehyde, m-fluorobenzaldehyde, 3,5-dihydroxybenzaldehyde, 3,5-difluorobenzaldehyde, m-mercaptobenzaldehyde, m-nitrobenzoic acid, m-hydroxybenzoic acid, 3,5-dinitrobenzoic acid, m-bromobenzoic acid, m-chlorobenzoic acid, m-fluorobenzoic acid, m-mercaptobenzoic acid, 3,5-dihydroxybenzoic acid, m-aminobenzoic acid and the like. Among them, m-hydroxybenzoic acid is preferred. These may be used singly or two or more of them may be used in combination.
In the above formula xe2x80x94NR2COxcfx862Y, R2 represents a hydrogen atom or a methyl or ethyl group. CO represents a carbonyl group. xcfx862 represents an atomic group comprising an aromatic ring bound to the nitrogen atom via said carbonyl group. COxcfx862Y represents an atomic group such that the corresponding compound represented by Yxcfx862COH has a logarithmic value logP of 0 to 3.2, P being the partition coefficient in the water-octanol system, (to the exclusion of p-nitrobenzoic acid).
If the logP value of the compound represented by the above HOCxcfx862Y is less than 0, the hydrophobic interaction with lipoproteins will be weak, hence the lipoprotein adsorbing ability will be low. If said logP value exceeds 3.2, not only LDL and VLDL but also HDL and other proteins will be adsorbed simultaneously, raising a problem from the selectivity viewpoint. The above range is thus critical. It is preferred that said logP value be 0.8 to 2.7.
While it is stated in Japanese Kokai Publication Sho-63-208764 that the hydrophobicity of the aromatic ring-containing atomic group Rb in the group represented by the above general formula xe2x80x94NRaRb plays an important role in the adsorption of lipoproteins, it has now newly been found that, from the ligand structure viewpoint, when said group Rb is bound via a CO bond, namely it is the above-mentioned xe2x80x94NR2COxcfx862Y, the selectivity for LDL is higher as compared with the cases where it is bound via a CS bond or CH2 bond and that when the ligand has a specific functional group Y, said selectivity is still higher.
Although the above-mentioned logP value is influenced by the particular functional group (e.g. methylene group, carbonyl group or the like) directly bound to the aromatic ring, said value will not vary much according to the presence or absence of such functional group or the kind thereof.
As the functional group Y, there may be mentioned, for example, halogens and hydroxy, acetyl, thiol, aldehyde, amino and like groups. The aromatic ring contained in the above group xcfx862 is not particularly restricted but may be any of the groups mentioned hereinabove in connection with the above xcfx861.
As the above group xe2x80x94NR2COxcfx862Y, there may be mentioned, in terms of the corresponding compound HNR2COxcfx862Y, such compounds as m-bromobenzamide, o-bromobenzamide, p-bromobenzamide, m-chlorobenzamide, o-chlorobenzamide, p-chlorobenzamide, m-fluorobenzamide, p-fluorobenzamide, o-iodobenzamide, p-iodobenzamide, m-nitrobenzamide, o-nitrobenzamide, p-nitrobenzamide, m-mercaptobenzamide, o-mercaptobenzamide, p-mercaptobenzamide, m-hydroxybenzamide, o-hydroxybenzamide, p-hydroxybenzamide, 2,4-dihydroxybenzamide, 2,5-dihydroxybenzamide, 2,6-dihydroxybenzamide, 3,5-dihydroxybenzamide, 3,4,5-trihydroxybenzamide, m-aminobenzamide, o-aminobenzamide, p-aminobenzamide and the like. Among these, those having hydoxy, nitro or fluoro as the functional group Y, namely m-hydroxybenzamide, 3,5-dihydroxybenzamide, m-nitrobenzamide, 3,5-dinitrobenzamide, m-fluorobenzamide, 3,5-difluorobenzamide and the like are preferred, and m-hydroxybenzamide, 3,5-dihydroxybenzamide, m-fluorobenzamide and 3,5-difluorobenzamide are more preferred. These may be used singly or two or more of them may be used in combination.
When the lipoprotein adsorbent of the present invention has the above-mentioned group xe2x80x94NR2COxcfx862Y, said adsorbent shows improved selectivity, namely the selectivity index thereof as represented by the formula (2) mentioned above is at least about 2.5 times higher as compared with the lipoprotein adsorbents described in Japanese Kokai Publication Sho-63-208764.
The nitrogen atom in the above group xe2x80x94NR2COxcfx862Y may be either of water-insoluble carrier origin or of ligand origin according to the method of introducing the above group xe2x80x94NR2CO xcfx862Y into the water-insoluble carrier. For example,
(1) when said xe2x80x94NR2COxcfx862Y group is a group derived from a compound represented by the general formula 
xe2x80x83(in which R2, CO, xcfx862 and Y are as defined above) (hereinafter expressed also as xe2x80x9cxe2x80x94HNR2COxcfx862Yxe2x80x9d) and immobilized on the water-insoluble carrier via the nitrogen atom in the compound mentioned above, the nitrogen atom in the above xe2x80x94NR2COxcfx862Y group is of ligand origin.
(2) When said xe2x80x94NR2COxcfx862Y group is introduced into a water-insoluble carrier by reacting the water-insoluble carrier having a group represented by the general formula 
xe2x80x83(in which R2 is as defined above) (hereinafter also expressed as xe2x80x9cxe2x80x94NR2Hxe2x80x9d) with a compound represented by the general formula Yxcfx862COOH (in which Y and xcfx862 are as defined above), the nitrogen atom in said xe2x80x94NR2COxcfx862Y group is of water-insoluble carrier origin.
As the compound represented by the formula HNR2COxcfx862Y, there may be mentioned those specifically given hereinabove.
The compound represented by xcex3xcfx862COOH is not particularly restricted but includes, for example, those compounds in which one or more groups each selected from the group consisting of halogens, hydroxy, acetyl, thiol, aldehyde and amino are bound to the aromatic ring of xcfx862. As specific examples, there may be mentioned m-bromobenzoic acid, o-bromobenzoic acid, p-bromobenzoic acid, m-chlorobenzoic acid, o-chlorobenzoic acid, p-chlorobenzoic acid, m-fluorobenzoic acid, p-fluorobenzoic acid, o-iodobenzoic acid, p-iodobenzoic acid, m-mercaptobenzoic acid, p-mercaptobenzoic acid, o-mercaptobenzoic acid, m-hydroxybenzoic acid, p-hydoxybenzoic acid, o-hydoxybenzoic acid, 2,4-dihydoxybenzoic acid, 2,5-dihydroxybenzoic acid, 2,6-dihydroxybenzoic acid, 3,5-dihydroxybenzoic acid, 3,4,5-trihydroxybenzoic acid, acetylbenzoic acid, acetylaminobenzoic acid, formylbenzoic acid and the like. Among them, m-hydroxybenzoic acid and p-hydroxybenzoic acid are preferred. These may be used singly or two or more of them may be used in combination.
It suffices for at least one of the above-mentioned xe2x80x94NR1xcfx861X group and xe2x80x94NR2COxcfx862Y group to occur on at least one part of the surface of the water-insoluble carrier according to the present invention, and two or more xe2x80x94NR1xcfx861X and/or xe2x80x94NR2COxcfx862Y groups may occur thereon.
The water-insoluble carrier to be used in the practice of the present invention may be an inorganic carrier; an organic carrier such as a carrier comprising a synthetic polymer or a polysaccharide; or a composite carrier comprising an organic carrier and an inorganic carrier. Considering the environment for lipoproteins occurring in the body fluid, a hydrophilic carrier is preferred and, further, the adsorption of substances other than the target substances, namely the so-called nonspecific adsorption should be as little as possible. As such carrier, there may be mentioned, for example, polysaccharides such as crosslinked agarose, crosslinked dextran, crosslinked cellulose, crystalline cellulose, crosslinked chitin and crosslinked chitosan; synthetic polymers such as styrene-divinylbenzene, crosslinked polyvinyl alcohol, crosslinked polyacrylates and crosslinked polyamides; inorganic carriers such as glass beads and silica gel; organic-inorganic composite carriers comprising inorganic carriers, e.g. glass beads, whose surface are coated with a polysaccharide or some other organic macromolecular compound; organicxe2x80x94organic composite carriers comprising organic carriers, comprising synthetic polymers, whose surface are coated with a polysaccharide; and the like.
Said water-insoluble carrier may have a functional group which can be used for an immobilization reaction. As such group, there may be mentioned amino, carboxyl, hydroxy, thiol, aldehyde, halogen, acidanhydride, amide, ester, epoxy, silanol and like groups. As the water-insoluble carrier having the above-mentioned xe2x80x94NR1H group among such functional groups, for instance, there may be mentioned, for example, water-insoluble carriers comprising materials originally having said xe2x80x94NR1H group, such as chitosan and the like; and water-insoluble carriers provided with said xe2x80x94NR1H group by introducing said group into originally amino-free water-insoluble carriers by activating the latter with cyanogen bromide, epichlorohydrin, 1,4-butanediol diglycidyl ether or the like, followed by reaction with a compound represented by the general formula H2NR1 (in which R1 is as defined above).
Preferably, said water-insoluble carrier is rigid.
The term xe2x80x9crigidxe2x80x9d in this specification means that when the water-insoluble carrier is packed uniformly into a cylindrical column and blood, serum or plasma or a dilution thereof or such liquid after pretreatment such as blood corpuscle removal or serum protein removal is passed through the column, said carrier has rigidness to such an extent that consolidation due to carrier deformation or the like will never occur.
If, when a column is packed with the adsorbent of the present invention and incorporated into an extracorporeal circulation circuit, consolidation of the adsorbent occurs during on-line therapeutic treatment, a sufficient rate of flow of body fluid will not be obtained any longer, whereby the treatment period has to be prolonged or it may even become impossible to continue the treatment. To avoid the consolidation of the adosorbent, it is preferred that the adsorbent have a sufficient mechanical strength, namely it be rigid.
The microstructure of the adsorbent of the present invention may be porous or nonporous. For obtaining a high LDL and VLDL adsorbing capacity per unit volume, however, it is preferred that the specific surface area be large, namely the adsorbent be porous, in particular wholly porous.
Said xe2x80x9cporousxe2x80x9d preferably means that the pore volume amounts to not less than 20% of the apparent volume of the water-insoluble carrier and the specific surface area amounts to not less than 3m2/g. Those carriers which fail to meet these requirements are not suited for practical use because of their limited adsorption capacity.
When said water-insoluble carrier is porous, it preferably has an exclusion limit molecular weight of 1 million to 100 million as measured using spherical proteins. When said water-insoluble carrier is porous, its exclusion limit molecular weight of not less than 1 million is used since it is necessary for LDL and VLDL molecules having a molecular weight of not less than 1 million to readily penetrate into pores of the water-insoluble carrier. If the exclusion limit molecular weight is less than 1 million, the adsorption capacity will be small, hence such carrier will be unsuited for practical use. If said exclusion limit molecular weight exceeds 100 million, the adsorbent will become weak in mechanical strength or the solid content of the adsorbent will be too low to have a sufficient adsorption capacity, hence will be unsuited for practical use. Thus, a preferred range is 1 million to 100 million and a more preferred range is 3 million to 70 million.
Said exclusion limit molecular weight is the molecular weight of the smallest molecule among molecules which are incapable of penetrating into pores in gel permeation chromatography, namely are excluded, as described, for example, in the monograph xe2x80x9cJikken Kosoku Ekitai Kuromatogurafi (Experiments in High Performance Liquid Chromatogarphy)xe2x80x9d (Hiroyuki Hatano and Toshihiko Hanai, published by Kagaku Dojin).
The porous carrier mentioned above is not particularly restricted but includes, for example, porous cellulosic carriers, porous chitosan carriers, vinylic porous carriers comprising styrene-divinylbenzene copolymers, crosslinked polyacrylates and crosslinked polyvinyl alcohol; and inorganic porous carriers such as glass, silica, alumina and the like.
As the method of producing the lipoprotein adsorbent of the present invention, there may be mentioned, for example, the method for introducing the above group xe2x80x94NR1xcfx861X by the method which comprises binding a compound represented by the above formula HNR1xcfx861X to a water-insoluble carrier via the nitrogen atom in said compound or reacting a water-insoluble carrier having the above-mentioned group xe2x80x94NR1H with a compound represented by the above Zxcfx861X, for instance, or method for introducing the above group xe2x80x94NR2COxcfx862Y by the method which comprises binding said compound represented by the formula HNR2COxcfx862Y to a water-insoluble carrier via the nitrogen atom in said compound or reacting water-insoluble carrier having the above-mentioned group represented by xe2x80x94NR2H with a compound represented by the above formula Yxcfx862COOH for instance. Further, there can be used the method which comprises introducing the above-mentioned group xe2x80x94NR1xcfx861X or xe2x80x94NR2COxcfx862Y into a water-soluble macromolecular compound and then subjecting the reaction product to crosslinking or like treatment to thereby obtain an adsorbent in the form of a water-insoluble form.
Said water-soluble macromolecular compound is not particularly restricted but includes, among others, polysaccharides such as dextran and starch; polymers such as polyvinyl alcohol and saponification products derived from ethylene-vinyl acetate copolymers with a low ethylene content; and the like.
The adsorbent obtained by the above production method has the above-mentioned xe2x80x94NR1xcfx861X or xe2x80x94NR2COxcfx862Y group introduced into the above-mentioned water-insoluble carrier or the above-mentioned water-soluble macromolecular compound. The method of immobilization is not particularly restricted but may be any of various known ones, such as physical, ionic or covalent bonding.
In the practice of the present invention, however, it is preferred that the ligand is immobilized on said water-insoluble carrier or water-soluble macromolecular compound in the manner of covalent bonding which affords little possibility of ligand leakage. Moreover, if necessary, a spacer may be introduced between the water-insoluble carrier and the ligand, namely the group xe2x80x94NR1xcfx861X or xe2x80x94NR2COxcfx862Y.
The form or shape of the adsorbent of the present invention is not particularly restricted but may be selected from such arbitrary shapes as granules, aggregates of particles, fibers, membranes and hollow fibers.
The adsorbent of the present invention can be used for removing LDL and VLDL from lipoprotein-containing liquids such as blood, serum or plasma, dilutions thereof, or liquids obtained therefrom after such pretreatment as blood corpuscle removal or serum protein removal. More specifically, it can be used as an adsorbent for the treatment of patients with hyperlipidemia or as an adsorbent for the analysis of various lipoproteins.
The adsorber of the present invention comprises an adsorption section containing the lipoprotein adsorbent of the present invention, a liquid inlet section for a liquid to flow into said adsorption section and a liquid discharge section for the liquid that has flown into said adsorption section to flow out of said adsorption section. It is preferred that said adsorber is provided with a filter which said liquid and components contained therein can pass through but the adsorbent of the present invention cannot pass through.
For using the adsorbent of the present invention for therapeutic purposes, various techniques are available. The simplest and easiest technique comprises pooling blood of a patient extracorporeally in a blood bag or the like, admixing the blood with the adsorbent of the present invention to remove LDL and VLDL, removing the adsorbent by passing through a filter and returning the blood to the patient. While this technique does not require any complicated apparatus, the amount of blood per one treatment is small. Moreover, it is time-consuming and troublesome.
Another technique comprises using an adsorber packed with the adsorbent of the present invention.
Specifically, an adsorber comprising a column packed with the adsorbent of the present invention is incorporated in an extracorporeal circulation circuit, and the adsorption and removal are effected on line mode. As said treatment, there can be mentioned the method which comprises circulating plasma whole blood directly through said circuit or the method which comprises separating plasma from blood and passing the plasma through said adsorber. The adsorbent of the present invention and the adsorber in which said adsorbent is used can be used for both modes of treatment.